Several obstacles exist for the obtaining of an effective vaccine against the HCV. Because its RNA nature, HCV can quickly mutate in adaptation to the environment. This contributes to the high diversity of sequences of the multiple viral isolates identified in the world. The biggest heterogeneity concentrates on the hypervariable region I of the HCV E2 protein, where a possible neutralizing epitope has been described. The HCV causes persistent infection in spite of the existence of an active immune response (Lechmann et al., Semin Liver Dis 2000, 20, 211-226). Neither an animal model, nor an in vitro culture system for the efficient replication of the virus and the study about the occurrence of neutralizing antibodies exist. The immunologic patterns associated with the progression of the illness or with the protection have not been defined. It is probable that potent, multispecific and long-lasting both, humoral and cellular immune responses are required for the resolution of the infection (Lechmann et al., Semin Liver Dis 2000, 20, 211-226).
Several approaches have been used to develop a vaccine against the HCV. Recombinant proteins, synthetic peptides, virus like particles, DNA vaccines and live-viral vectors are the most widely evaluated.
The development of a vaccine based on protein subunits was one of the first strategies evaluated for the HCV because for several flaviviruses, antibodies directed against surface proteins can confer protection. Some variants based on the HCV structural antigens have achieved limited protection against the virus in animal models. Such it is the case of the chimpanzees immunized with E1-E2 heterodimers. Seven chimpanzees were vaccinated, five were protected and two developed a self-limiting disease (Choo et al., PROC NATL ACAD SCI USES 1994, 91, 1294-1298). This protection has been correlated with the presence of antibodies (Abs) able to inhibit the E2 binding to human cells (Rosa et al., PROC NATL ACAD SCI USES 1996, 93, 1759-1763).
The recombinant E1 protein from an isolate of the genotype 1 b was purified as homodimers self-associating in particles of 9 nm diameter, approximately (Maertens et al., Records Gastroenterol Belg 2000, 63, 203). Two chimpanzees chronically infected with HCV received 9 doses of 50 μg of the recombinant E1 protein. The vaccination improved the hepatic histology and determined the disappearance of the viral antigens of the liver. Vaccination with recombinant E1 protein also reduced the levels of alanine aminotransferase (ALT). Although the levels of viral RNA in serum didn't change during the treatment, the liver inflammation and the levels of viral antigens increased after treatment. An association was observed between the high levels of antibodies against E1 and the improvement of the illness (Maertens et al., Records Gastroenterol Belg 2000, 63, 203).
Particularly, the formation of virus-like particles from recombinant proteins and their employment as vaccines is very attractive because these structures frequently simulate viral properties. This kind of particles, obtained from insect cells infected with a recombinant baculovirus containing the sequence of the HCV structural antigens, have been able to generate both humoral and cellular immune response against these antigens (Baumert et al., Gastroenterology 1999, 117, 1397-407; Lechmann et al., Hepatology 1999, 30, 423-429). Although the results obtained with vaccines based on protein subunits are encouraging, the immune response induced by these variants is mainly humoral, short-term and isolate-specific.
On the other hand, different recombinant viral vectors have been evaluated in the development of a recombinant vaccine against the HCV. Particularly, recombinant adenoviral vectors are interesting candidates due to their liver tropism, their power to induce both humoral and cellular immunity, and the feasibility for oral or systemic delivery. Adenoviruses containing the DNA encoding sequence for the HCV structural proteins induce an antibody response against each one of these proteins (Makimura et al., Vaccine 1996, 14, 28-36). Moreover, after immunization in mice with recombinant adenoviruses for C and E1, a specific CTL response is detected against these antigens (Bruna-Romero et al., Hepatology 1997, 25, 470-477). Although these results have been encouraging, the recent problems with the use of recombinant adenoviruses in gene therapy have raised doubts about their employment in humans. Other recombinant viruses, like vaccinia, canary-pox and fowl-pox, containing different HCV genes have induced strong CTL and T-helper immune responses in mice (Shirai et al., J Virol 1994, 68, 3334-3342; Large et al., J Immunol 1999, 162, 931-938). However, these recombinant viruses, as well as other variants of alpha virus like the Semliki Forest Virus are also affected by regulatory issues and security concerns related with their application.
The identification of several epitopes for CD4+ and CD8+ T cells in the HCV polyprotein, which could be important in the viral elimination, support the evaluation of synthetic peptides as vaccine candidates against this pathogen. Different peptides, lipidated or not, containing epitopes of C, NS4 and NS5, have induced a strong CTL response in mice (Shirai et al., J Infect Dis 1996, 173, 24-31; Hiranuma et al., J Gene Virol 1999, 80, 187-193; Oseroff et al., Vaccine 1998, 16, 823-833).
Another strategy used to develop a vaccine against the HCV is based in the possibility of generating Abs against linear epitopes. This alternative has been evaluated basically to generate Abs against the HVR-I of the HCV, with some encouraging results in rabbits and chimpanzees (Esumi et al., Arch Virol 1999, 144, 973-980; Shang et al., Virology 1999, 258, 396-405). Quasi-species is the main problem of selecting the HVR-I as the target for a vaccine against the HCV.
The main obstacle for the peptide vaccines is that those peptides without epitopes for helper T cells can be poorly immunogenics. Moreover, the effectiveness of a vaccine is frequently based on the induction of specific immune response against a wide range of different antigens. These limitations are important weaknesses of this strategy.
The DNA immunization is one of the most recent strategies in vaccine development. A DNA vaccine consists on a purified plasmid containing the sequence coding for an antigen of interest, under the control of a functional transcriptional unit in eucariotic cells. After injection of the plasmid in muscle or the skin, the plasmid is taken up by host cells and the antigen is expressed intracellularly. The expression of the encoded antigens in the host cells is one of the major advantages of this methodology because is similar to viral natural infections. The simplicity to manipulate the DNA, together with the DNA stability that makes possible a relatively cheap large-scale production of DNA, is other advantage of DNA vaccination.
The immune response induced with this kind of vaccines can be modulated by co-immunization with molecules or genes coding for co-stimulatory molecules like cytokines. The genetic constructs can be modified, by insertions or deletions of transmembrane domains, signal sequences for secretion, or other types of residues affecting the intracellular trafficking and processing of the antigen.
Particularly, the DNA immunization has been largely studied in the development of vaccines against the HCV. Different expression vectors encoding full-length or truncated variants of the HCV capsid protein have been generated (Lagging et al., J Virol 1995, 69, 5859-5863; Chen et al., Vaccine Res 1995, 4, 135-144). Other constructs also include the HCV 5′ non-translated region (Tokushige et al., Hepatology 1996, 24, 14-20). Plasmids expressing fusion variants to the hepatitis B virus (HBV) surface antigen or other envelope antigens of the HBV have been evaluated (Major et al., J VIROL 1995, 69, 5798-5805). Immunization with these plasmids has generally induced positive CTL and lymphocyte proliferative response.
The HCV envelope proteins have also constituted targets of interest for this type of technology. In the case of the HCV E2, the humoral response seems to be mainly directed to the HVR-1 (Lee et al., Mol Cells 1998, 8, 444-451). Immunization with plasmids expressing intracellular or secreted variants of the E1 and E2 proteins has rendered similar immune response (Lee et al., J VIROL 1998, 72, 8430-8436). The inoculation with bicistronic plasmids expressing the GM-CSF and the HCV E1 or E2 proteins increased both humoral and cellular immune response. Recently, the use of bicistronic plasmids expressing the E1 and E2 proteins were generated to investigate the influence of heterodimer formation between these proteins in vivo on the immune response induced after DNA immunization. When heterodimers were formed, the antibody response against HCV E1 and E2 proteins was not obtained. In sharp contrast, high-level antibody titers, directed to both linear and conformational epitopes, were obtained after immunization with plasmids expressing truncated variants of E1 and E2. Therefore, it seems necessary to avoid the heterodimers formation to obtain a strong antibody response when constructs including these antigens are evaluated (Fournillier et al., J VIROL 1999, 73, 497-504).
The non structural proteins have also been evaluated by this technology. Good results were obtained when the region coding for the C-terminal domain of the NS3 protein was included in a vector that allows the simultaneous and independent expression of this domain and the IL-2 (Papa et al., Res Virol 1998, 149, 315-319). The NS4 and NS5 proteins also generate Abs and CTL responses by this immunization strategy (Encke et al., J IMMUNOL 1998, 161, 4917-4923). Recently, the use of a construction coding for GM-CSF and the non structural proteins of the virión (NS3, NS4 and NS5) induced a potent Ab response and a potentiated lymphoproliferative response against each non structural protein (Cho et al., Vaccine 1999, 17, 1136-1144).
In general, the effective expression of different HCV antigens, as well as the generation of anti-HCV Abs in levels ranging from 1:100 to 1:100 000 according to the combination in study, has been reported for different DNA constructs (Inchauspe et al., Vaccine 1997, 15, 853-856). Additionally, the development of specific CTL and lymphocyte proliferative response has been demonstrated (Inchauspe et al., DNA AND CELL BIOLOGY 1997, 16, 185-195). However, efforts are required to improve this methodology in order to generate stronger both humoral and cellular response against different proteins of the HCV. Thus, some variants like liposomes (Gramzinski et al., Mol Medicine 1998, 4, 109-118) and saponin QS-21 (Sasaki et to the., J Virol 1998, 72, 4931-4939) have been evaluated to increase the immune response induced after DNA vaccination. The dendritic cells as biological adjuvants have been also studied in DNA immunization. Dendritic cells (CD) derived of former genetically modified mouse bone marrow to express tumor antigens, by using viral vectors (Specht et al., J Exp Med 1997, 186, 1213-1221; Brossart et al., J Immunol 1997, 158, 3270-3276; Song et al., J Exp Med 1997, 186, 1247-1256), or RNA (Boczkowski et to the., J Exp Med 1996, 184, 465-472), have demonstrated their capacity to promote T cell response specific for tumor antigens, and prophylactic immunity mediated by cells against tumors in mouse.
At the present, the improvement of vectors for DNA immunization, including the insertion of CpG motifs to increase the immune response against the expressed antigens (Hasan et al., J Immunol Meth 1999, 229, 1-22), and the DNA delivery systems is crucial to overcome the limitations of this technology. Due to the challenges that outlines the HCV infection, and to the absence of a clear definition about the immunologic parameters correlating with the protection against this pathogen, it is possible that an effective vaccine against the HCV shall require a multispecific approach stimulating several aspects of the immune response. The solution of this problem is probably in the combination of several vaccination strategies explored until the moment. Particularly, immunization schedules that combine a prime dose with a DNA vaccine and a booster dose with recombinant proteins or viral vectors (Hu et al., Vaccine 1999, 17, 3160-3170; Pancholi et al., J Infect Dis 2000, 182, 18-27) have been evaluated with results that, although positives, require additional investigations to demonstrate if the prime-boost strategies can really induce a protective immunity against the HCV.
Additionally, for the hepatitis B model, a vaccine composition comprising the complex formed by the hepatitis B surface antigen, an antibody specific for this antigen, and a DNA vaccine expressing for this antigen has been evaluated (Wen et al., U.S. Pat. No. 6,221,664, 1998). This formulation allowed the antigen presentation by different means and a quick induction of immune response that resulted superior regarding to the one generated by the individual variants.